The presence of an abnormally high level of leukocyte cells in the urine of an individual is indicative of a pathological condition, such as a kidney or urogenital tract infection. The detection of leukocyte esterase in urine is an indirect test for bacteriuria (i.e., an abnormally high level of bacteria), and therefore infection. Accordingly, an accurate urine leukocyte cell assay, or leukocyte esterase assay, is valuable to a physician in the diagnosis and treatment of kidney and urogenital tract infections.
Traditionally, technicians relied upon visual techniques to count leukocyte cells either in urine sediment or in uncentrifuged urine. The conventional visual technique requires expensive equipment, such as a centrifuge and microscope, in addition to an inordinate amount of technician time. An additional disadvantage of the visual technique is that only intact leukocyte cells can be counted. However, leukocyte cells in the urinary system are subject to extensive cell lysis. For example, in a urine having an abnormally high pH, the half life of a leukocyte cell can be as low as 60 minutes. Since lysed leukocyte cells escape detection in the visual technique, an erroneously low, or a false negative, result for leukocyte cells can result.
A visual examination for leukocyte cells in urine can be performed on uncentrifuged urine or on urine sediment. The latter method requires centrifuging the urine sample, isolating the sediment, then visually inspecting the sediment, wherein the technician counts the number of leukocyte cells appearing in the viewing field. This visual technique is complicated by the presence of other components in the urine sediment, such as epithelial cells and salt particles. The presence of various urine sediment constituents, coupled with other factors, like a nonhomogeneous sample or differing optical powers between microscopes, can lead to substantial assay errors.
Therefore, a quick, easy method of assaying for leukocyte cells, which eliminates the need for time-consuming counting techniques and expensive equipment, and which provides an accurate assay for intact and lysed leukocyte cells, would constitute a significant advance in the art. The present invention provides such an advance. Moreover, because the present invention is based on the enzymatic activity of esterase or protease in leukocyte cells, and not on the ability to visually observe and count intact leukocyte cells, the method is free of the assay inaccuracies described above.
Prior to the present invention, compositions and methods of determining the presence or concentration of leukocyte cells, esterase or protease in a test sample utilized chromogenic esters which produced an alcohol product as a result of hydrolysis by an esterase or protease. The intact chromogenic ester has a color different from the alcohol hydrolysis product. The color change generated by hydrolysis of the chromogenic ester therefore provides a method of detecting the presence or concentration of esterase or protease, which in turn is correlated to the presence or concentration of leukocyte cells. Many of these prior compositions used accelerator compounds and diazonium salt coupling agents in conjunction with the chromogenic ester to improve assay response.
A present day urine assay for leukocyte esterase, and therefore indirectly for leukocyte cells, is a dry phase test strip termed LEUKOSTIX.RTM., available from Miles, Inc., Elkhart, Ind. This test strip detects esterase activity released from granules of leukocyte cells into the urine. The released esterase is termed "human leukocyte elastase" (HLE), and is a serine protease-based enzyme.
In the assay for leukocyte esterase or HLE using a LEUKOSTIX.RTM. strip, the enzyme hydrolyzes a--chromogenic ester incorporated into the strip to form a pyrrole compound, which in turn reacts with a diazonium salt to form a highly colored azo dye. The degree and intensity of the color transition is proportional to the amount of leukocyte esterase or HLE in the urine, which in turn, is proportional to the number of leukocyte cells in the urine.
The reaction chemistry of the LEUKOSTIX.RTM. test strip is illustrated as follows: ##STR3##
In particular, the LEUKOSTIX.RTM. assay for leukocyte esterase is based on the splitting, i.e., hydrolysis, of the 3-hydroxy-5-phenyl-pyrrole-N-tosyl-L-alanine ester (I) by an enzyme or enzymes to form 3-hydroxy-5-phenyl-pyrrole (II). The hydroxy-pyrrole (II) then reacts with a diazonium salt (III) (e.g., 1-diazo-2-naphthol-4-sulfonic acid) to produce azo dye (IV) having a purple color.
The reacted LEUKOSTIX.RTM. test strip is matched to a color chart having four color blocks of increasing color intensity from trace to 3+, which in an average urine represents a leukocyte cell concentration of about 10 to greater than 500 cells/.mu.L (microliter), or 30 to greater than 1500 ng/mL (nanograms per milliliter) of HLE. In low specific gravity test samples, even smaller concentrations of HLE can be detected.
The intensity of the color is proportional to the amount of enzyme (e.g., HLE) present in the urine sample and, therefore, is directly related to the number of leukocyte cells in the urine. An assay which generates a color of 1+ or more is a definite indication that a significant number of leukocyte cells are present in the urine sample.
The present day composition and method of assaying for leukocyte cells is disclosed in Corey et al. U.S. Pat. No. 4,657,855 and Skjold et al. U.S. Pat. No. 4,637,979. As disclosed therein and as discussed above, the current LEUKOSTIX.RTM. test strips rely upon an amino acid ester, and in particular an alanine ester, to provide a color transition in the assay for leukocyte cells. Other patents related to assaying for an esterase or protease enzyme include Hugl et al. U.S. Pat. Nos. 4,806,423 and 4,814,271.
In contrast to present-day compounds and compositions used to assay for HLE and leukocyte cells, the method and test device of the present invention utilize a lactate ester, like a hydroxy-protected 5-phenyl-3-hydroxy-pyrrolyl-L-lactate. The present lactate esters have increased reactivity relative to the corresponding alanine esters, such as the N-tosyl alanine ester having structural formula (I) depicted above. A combination of the present lactate esters and a diazonium salt coupling agent also provide a more sensitive assay for HLE. The lactate esters, like the alanine esters, undergo a detectable or measurable response, like a color transition, upon contact with leukocyte cells, esterase or protease. The response is proportional to the concentration of leukocyte cells, esterase or protease in a test sample.
Other patents and publications disclose the hydrolysis of indoxyl- and thioindoxyl-alanine esters to generate a color transition or other detectable response. These references include GB Patent No. 1,128,371; Janoff et al., Proc. Soc. Exper. Biol. Med., 136, pp. 1045-1049 (1971); Sweetman et al., Jour. Hist. Soc., 22, pp. 327-339; and Berger et al. U.S. Pat. No. 4,278,763.
To date, no known patent or publication discloses the use of a lactate ester as an enzyme substrate in the assay for leukocyte cells, esterase or protease. Dorn et al. U.S. Pat. No. 4,064,236 and Dorn et al., J. Med. Chem., 20, pp. 1464-1468 (1977) disclose inhibitors for pancreatic and granulocyte elastase. Some of the compounds contain a lactate moiety at the non-scissle and scissle points of cleavage. These compounds range from being strong to very weak inhibitors. These compounds do not contain activated leaving groups and are designed primarily as inhibitors to elastase with carbazate esters resembling alanine present at non-hydrolytic sites to increase selectivity for inhibiting elastase. Similarly, Japanese Kokai JP 52/057,121 discloses lactoyl-polypeptide compounds exhibiting potent antipepsin and anticathepsin inhibitory activity, wherein the lactate moiety again is incorporated in an inhibitory capacity. In contrast, the present lactate esters are chromogenic substrates having high esterase (HLE) enzymatic activity, and the ester linkage of the chromogenic lactate ester is the hydrolysis site.
J. Dufer et al., Ann. Pharm. Fr., 31, pp. 441-450 (1973) discloses a 9-methoxyellipticine lactate salt having a decreased esterase activity in lymphocytes, and which was used against acute myeloblastic leukemia. The disclosed lactate salt is used as a solubilizing component for the ellipticine, and has an inhibitory activity for esterase.
H. Moorlag et al., J. Org. Chem., 55, pp. 5878-5881 (1990) and H. Moorlag et al., Tetrah.; Assym., 2, pp. 705-720 (1991) disclose the enzymatic hydrolyses of a variety of .alpha.-substituted mandelic esters, .alpha.-substituted lactic esters and racemic .alpha.-substituted .alpha.-hydroxy esters using pig liver esterase to determine enantioselectivity. Pig liver esterase showed no enantioselectivity for the .alpha.-substituted lactic esters.
F. Kraicsovits et al., Symp. Pap. IUPAC Int. Symp. Chem. Nat. Prod., 1, pp 37-40 (1978) discloses the effect of structure on the reactivity of substrates in the presence of the serine protease, chymotrypsin, wherein particular non-amino acid substrates contained the lactate moiety. In these examples, the lactate moiety is not at the hydrolysis site, but rather one residue removed from the hydrolysis site.
Publications such as J. W. Harper et al., Biochem., 23, pp. 2995-3002 (1984); G. Digenis et al., J. Med. Chem., 29, pp. 1468-1476 (1986); A. Krantz et al., J. Med. Chem., 33, pp. 464-479 (1990); and D. W. Ingles et al., Biochem. J., 108, pp. 561-569 (1968) disclose that the natural substrate at the HLE cleavage site is an amino acid, such as alanine or valine.
In particular, the D. W. Ingles et al. publication discloses rates of deacylation of acyl-.alpha.-chymotrypsins wherein the nitrogen-hydrogen bonding capacity of the acylamino group of the substrate has been eliminated by replacing the nitrogen with an oxygen (i.e., conversion to a lactate). When L-phenyl-alanyl was changed to L-phenyl-lactyl, the substitution resulted in a ten fold decrease in the enzymatic rate. Stereospecificity also decreased by a magnitude of up to 700 in replacing the amino NH moiety with a lactate OR moiety, wherein R is acetate or carboxyphenyl.
Other publications disclosing the hydrolysis of lactates and/or alaninates include:
J. Suh et al., J. Am. Chem. Soc., 107, pp. 4530-4535 (1985);
J. Suh et al., J. Am. Chem. Soc., 98, pp. 1940-1947 (1976);
J. Sub et al., Biochemical and Biophysical Research Communications, 64, pp. 863-869 (1975);
L. C. Kuo et al., J. Mol. Biol., 163, pp. 63-105 (1983);
S. J. Hoffman et al., J. Am. Chem. Soc., 105, pp. 6971-6973 (1983);
P. L. Hall et al., J. Am. Chem. Soc., 91, pp. 485-461 (1968); and
M. W. Makinen et al., Proc. Natl. Acad. Sci. USA, 73, pp. 3882-3886 (1976).
Therefore, in order to detect the onset and to monitor the progression of a kidney or urogenital tract infection, an accurate and sensitive assay.of urine for leukocyte cells, esterase or protease is needed for both laboratory and home use. The assay should permit the detection and measurement of the leukocyte cells, esterase or protease in the test sample such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained. In addition, it would be advantageous if the assay method utilizes a dry phase test strip in a dip-and-read format for the easy and economical determination of leukocyte cells or HLE in urine.
Present day test strips for leukocyte cells need improvement in the areas of sensitivity and speed of assay. Therefore, it would be a significant advance in the art of diagnostic assays if test strips for leukocyte cells were more sensitive to low concentrations of leukocyte cells and provided assay results in about 60 seconds. It was towards achieving these improvements that investigations resulting in the composition, device and method of the present invention were directed.
The method of the present invention provides a fast, accurate and trustworthy assay for leukocyte cells, esterase or protease by utilizing a test strip having a test pad comprising a suitable carrier matrix incorporating a reagent composition of the present invention. The reagent composition comprises a lactate ester, and in particular, a hydroxy-protected 5-phenyl-3-hydroxy-pyrrolyl-L-lactate. The composition further comprises a buffer, and optionally can include an accelerator compound and/or a diazonium salt coupling agent. The reagent composition is sensitive to trace concentrations of leukocyte cells, esterase or protease. The present reagent composition enhances the sensitivity of the assay for leukocyte cells, esterase or protease, thereby providing a more accurate and trustworthy assay.
No known method of assaying urine or other test samples for leukocyte cells, esterase or protease used a reagent composition comprising a lactate ester of the present invention.